Process for inhibiting enzymatic activity

ABSTRACT

A process for inhibiting enzymatic activity in a substrate in liquid phase by the application of a denaturing means to the substrate up to a temperature set between 0 and 60° C., preferably set between 10 and 50° C., and more particularly at room temperature. As the denaturing means either the application to the substrate of gaseous ozone or the application to the substrate of UV waves for a predetermined time are provided. UV waves and gaseous ozone may also be provided as a combined denaturing means, at the same time or in different times, generated by the same device. Examples of the substrate to which the invention is applied can be: a food matrix, such as vegetable juice or puree, fruit juice or puree; a pure enzyme or a mixture of enzymes in liquid phase, in particular contained in liquid food; enzymes and mixture of reagents used for biochemical synthesis; and waste material where inhibiting the enzymatic activity is necessary before disposal. The process does not damage or affect the substrate and do not apply heat, and successfully inhibits the enzymatic reactions caused by enzymes present in the substrate. The advantage of working at a temperature less than 60°, and preferably at room temperature, implies less energy absorption since the substrate must not any more be heated and cooled as required by traditional techniques, as well as it does not affect the characteristics, vitamins and fragrances of the treated food substrates.

FIELD OF THE INVENTION

The present invention describes methods suitable for inhibitingenzymatic activity in biochemical industry where in general enzymaticreactions have to be controlled and especially in food, cosmetic, andpharmaceutical industries.

More particularly, the invention relates to a process for inhibitingenzymatic activity during the production of puree and juice food.

BACKGROUND OF THE INVENTION

Enzymes are complex protein molecules that carry out precise catalyticfunctions within certain biochemical reactions. They are sequences ofmonomer units consisting of amino acids but also have an essentialsecondary and tertiary structure (three-dimensional). Such a structuregives to enzymes a considerable catalytic activity with respect to manychemical reactions that occur in living organisms. Normally, anenzymatic reaction takes place in the following way: an enzyme combineswith a reagent, i.e., said substrate, forming an enzyme-substratecomplex, which then changes into an enzyme-product complex and in turnsplits into product and enzyme free from each other, ready to react withanother molecule of substrate.

The process may take place very quickly; in many cases a single moleculeof enzyme is capable of transforming into product in a very short timethousands of molecules of substrate. A reaction catalyzed by an enzymecan be up to 1000 times quicker than the same reaction but notcatalyzed.

In biochemical industry enzymatic reactions play a primary role and inparticular are used to control and sometimes inhibit enzymatic reactionsafter that they have completed their task or, simply, when they areundesired.

In the food industry, in certain known production processes of fruitjuice, for example, some enzymatic reactions are used among whichpeptization of pectin where suitable enzymes such as pectinase areadded. Such an enzymatic digestion allows a complete extraction of thejuice and to prevent it from mucilage growth.

In many other cases, always in the food industry, properly inhibitingenzymatic activity allows not only an appropriate processing but also along food conservation for example of vegetable or animal puree, fruitjuice, syrups and other types of liquid food, for example, tomato juiceand milk.

A common technique for inhibiting enzymatic activity, especially in thefood industry, consists of pasteurization. Pasteurization providesheating the food substrate (either juice or fruit or other) up to atemperature set between 60 and 90° C. and more for a time variableaccording to the substrate to treat. For example, tomato juice heated upto 121° C. for 0.7 minutes for inhibiting Bacillus coagulans[Kirk-Othmer Encyclopaedia of Chemical Technology 3rd Ed. J. Wiley &Sons, Vol. 11, p. 300]. In fact, heating can cause permanentmodifications in the secondary or tertiary structure of the proteinsthat make up the enzymes such that their catalytic activity is stopped,causing a denaturation and then inhibiting the enzymatic activity.

Concerning fruit, a process for inhibiting enzymatic activity by heatingis described in EP0850572, relative to vegetable or animal puree,capable of giving the product a controlled exposition to heat.

Inhibiting enzymatic activity by heating, however, can be used onlywhere a short heating does not damage or affect too much the foodsubstrate. In any case, the application of heat to a food substrate, forexample a juice or a vegetable or animal puree, changes the organolepticfeatures and destroys partially or completely thermo-sensitive vitamins.

SUMMARY OF THE INVENTION

It is therefore a feature of the present invention to provide a processfor inhibiting enzymatic activity that does not damage or affect toomuch the substrate.

It is another feature of the present invention to provide a process forinhibiting enzymatic activity that do not apply heat.

According to one exemplary embodiment of the invention, a process isprovided for inhibiting enzymatic activity in a substrate in liquidphase in order to stop or inhibiting the course of enzymatic reactionscaused by enzymes present in said substrate, the process comprising thestep of denaturing the enzymes by the application of a denaturing meansto the substrate up to a temperature set between 0 and 60° C.,preferably set between 10 and 50° C., and more particularly at roomtemperature.

In a first exemplary embodiment the denaturing step provides theapplication to the substrate of gaseous ozone as the denaturing means.

In a second exemplary embodiment the denaturing step provides theapplication to the substrate of UV waves as a denaturing means for apredetermined time.

The application may also be provided to the substrate of UV waves and ofgaseous ozone as a combined denaturing means, at the same time or atdifferent times. In this case the combined denaturing means can begenerated by a same device that is both a source of UV waves and anozone generator.

The step of application to the substrate of UV waves is carried out at apredetermined frequency and intensity where said UV waves produce ozonein said substrate.

The substrate to which the invention is applied can be selected from thegroup consisting of a food matrix; a pure enzyme or a mixture of enzymesin liquid phase; enzymes contained in fruit juice or other liquid foodin a real matrix; enzymes and mixture of reagents used for biochemicalsynthesis; and waste material where inhibiting the enzymatic activity isnecessary before disposal.

The food matrix can be selected from the group consisting of vegetablejuice, vegetable puree, fruit juice, and fruit puree.

According to preferred embodiments of the invention, radiating with UVwaves or treating with ozone or the combination of the two treatmentsallows denaturing enzymes and then inhibiting the enzymatic activity infood substrates at room temperature.

The advantage of working at room temperature implies less energyabsorption since for example fruit juice must not any more be heated andcooled as the traditional techniques require. Furthermore, thetreatments proposed with the present invention, acting at a temperatureless than 60°, and preferably at room temperature, do not affect thecharacteristics, vitamins and fragrances of the treated food substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to some notlimitative examples with reference to FIGS. 1, 2 and 3, wherein:

FIG. 1 shows a hydrolysis chart of saccharose into glucose and fructoseby means of invertase enzyme and the effect of denaturing the enzyme bymeans of UV waves;

FIG. 2 shows a hydrolysis chart of saccharose into glucose and fructoseby means of invertase enzyme and the effect of denaturing the enzyme bymeans of UV waves at an intensity higher than the case of FIG. 1;

FIG. 3 shows a hydrolysis chart of saccharose into glucose and fructoseby means of invertase enzyme and inhibiting the latter by means ofozone.

DESCRIPTION OF THE PREFERRED EXEMPLARY EMBODIMENTS

In order to show the effectiveness of inhibiting enzymatic activityaccording to the invention, a model enzymatic reaction is selected andthe effect is studied of physical treating (UV irradiation) or chemicaltreating (treatment with ozone) standard saccharose solutions inparticular the reaction of saccharose inversion. Saccharose is a commontable sugar and is a disaccharide consisting of the union of a moleculeof glucose with one of fructose. The invertase enzyme (also calledsaccharase) is a protein that is obtained for example by thesaccaromyces cerevisiae micro organism, and consists of a mixture of twoproteins, α-glicoxydase and β-h-fructoxydase. Invertase enzyme is ahydrolase that is capable of driving the reaction of fission ofsaccharose into two components, i.e., glucose and fructose. Thisenzymatic reaction can be carried out in aqueous phase in many realsubstrates, for example, in fruit juice. In the present example thereaction has been made using standard solutions of saccharose. Thereaction of fission of saccharose into glucose and fructose is called areaction of sugar inversion, since saccharose has optical activity andhas the ability of rotating the plane of polarized light with a[α]_(D)=+66.5, whereas the products of the reaction, i.e., the glucoseand the fructose, have respectively a [α]_(D)=+52,5 and [α]_(D)=−92,09.Thus, at the end of the reaction the products will have globally a valueof [α]_(D)=−20,2. Since the reaction has a reagent at start that canrotate the plane of polarized light in a positive direction and forms amixture of products that instead rotate the plane of polarized light ina negative direction, the reaction is known as sugar inversion. Thereaction of inversion can be then followed easily by a polarimeter as isknown to those skilled in the art.

Examples on Experimental Substrates Example 1

A 10% solution of saccharose (200 ml) at room temperature having a 6,50°starting rotation of polarized light α, acidified with 8 droplets of 90%acetic acid to which 65 mg of invertase enzyme (Fluka) are added andstirred to dissolve/disperse the enzyme. 150 ml of a mixture of thereagents are put immediately after mixing in a photochemical reactorhaving a source of UV light emitted from a low pressure 12 W mercuryvapour lamp. The solution is immediately irradiated with UV and a flowof N₂ is insufflated in the solution under irradiation. The remainder 50ml of the starting solution are not irradiated and are used as “white”or reference sample. The reaction of inversion is followed at apolarimeter both for the irradiated solution with UV and for the “white”sample. The results are given in the chart of FIG. 1.

From the chart of FIG. 1 it can be understood that in the first 1,000seconds of irradiation there are not relevant effects in the kinetics ofthe inversion reaction. However after 15 minutes a considerabledeceleration in the reaction speed of the irradiated solution occurredwhereas the solution not irradiated has a normal kinetic course. If,after 17 min the irradiation ceases the kinetics of the reaction isdescribed by light dots, whereas, if irradiation continues also after 17min the kinetical trend is shown by dark dots. The comparison betweenlight dots and dark dots shows that already at 17 min of irradiation theenzymatic reaction is remarkably inhibited with respect to the “white”,but prolonging the irradiation (dark dots) after 17 minutes impliesinhibiting the enzymatic activity more completely.

Example 2

The same test described in Example 1 was repeated exactly in the abovedescribed conditions, but omitting insufflation with nitrogen in theirradiated solution and carrying out irradiation in static air. Theeffect has practically equivalent results. The example shows that theirradiation in nitrogen or in air is equivalent for invertase enzyme.

Example 3

The same test described in Example 1 was repeated with nitrogen on asolution of saccharose having a rotation starting value of polarizedlight α=8.35°, using a photochemical reactor having a UV source of 125 Wat medium-high Hg pressure instead of the 12 W source of the previousexamples. Even in this case the reaction of inversion is trackedpolarimetrically in comparison with a “white” sample of not irradiatedsolution. The results are shown in the chart of FIG. 2.

Using a more powerful source of UV light as the 125 W lamp atmedium-high Hg pressure, the inhibition on the invertase enzyme is clearin less than 10 minutes as can be seen by the trend of the inversionreaction kinetics (dark dots) in comparison with the not irradiatedreference reaction kinetics (triangles). The inhibiting action of the125 W lamp is complete and definitive with respect to that of examples 1and 2. If the irradiation is discontinued after 19 minutes (rhombs), theinversion reaction kinetics is in any case inhibited and the same occursin a solution irradiated for 85 minutes (dark dots). Even in this case,the inhibiting action of UV light on the invertase enzyme and then onthe inversion reaction is evident, clear and distinct.

Example 4

A 10% solution of 200 ml of saccharose in water having a startingrotation value of polarized light α=7.20° is put in 4 different vesselscontaining 50 ml each of starting solution, at a temperature of 25° C.

Solution 1 is not treated with ozone.

Solution 2 is treated with O₃ up to a nominal concentration of about 50mg O₃/litre insufflating air containing ozone at 17%.

Solution 3 is treated with O₃ up to a nominal concentration of 12 mgO₃/litre in the same way as for Solution 2.

Solution 4 is treated with O₃ up to a nominal concentration of 3 mgO₃/litre in the same way as for Solution 2.

Once ready, to all the Solutions 1-4, 48±2 mg of invertase enzyme havebeen added. The reaction kinetics were followed polarimetrically. Theresults are shown in the chart of FIG. 3. From the chart it is clearthat Solution 1, corresponding to the “white” sample (triangles), issubject to a normal reaction of inversion. Solution 4 treated with 3 mgO₃/litre shows only a slight delay in the reaction kinetics with respectto the “white” sample, demonstrating that at that concentration ozonehas not a significant inhibiting effect on invertase enzyme. However,Solutions 2 and 3 respectively 50 and 12 mg O₃/litre show instead a fullinhibition of the inversion reaction of saccharose. Particularlyinteresting is the result obtained with only 12 mg O₃/litre that appearsto be a minimum threshold from which it is possible to inhibitcompletely the action of invertase enzyme.

Examples on Real Substrates

Examples 1-4 have shown that both UV radiation and ozone carry out adenaturation capable of inhibiting completely the action of theinvertase enzyme.

UV radiation is a physical means suitable essentially for treatment oflimpid substrates and in any case of substrates that are transparent tothese waves. If the substrates are opaque a treatment is possible ofirradiation of a thin film of the substrate same. The UV radiation isabsorbed by the proteins and the denaturing effect is shown by aplurality of reactions (reticulations, degradations, isomerisms) thataffect both the primary structure of the enzymatic protein and thesecondary and tertiary structures. A minimum, but permanent, alterationof the structure of the protein causes necessarily a denaturation of theenzyme that then is permanently inhibited from driving a certainreaction.

The ozone instead is a chemical means and is a powerful oxidant.Surprisingly, it is very effective and safe in inhibiting the enzymaticactivity and it can applied in case of all food substrates, also opaqueor heterogeneous substrates, provided it is distributed suitably inthem. Ozone, then, is instable and slowly decomposes spontaneously intooxygen whereby its concentration becomes void in the treated substratesin a few hours.

In the following examples the results of some experiences are shownconcerning the effect of the ozone on real substrates. Fruit juice andfruit puree have been used, even if the examples are to be intended notlimitative for the extent of the invention, which can be applied to adesired food substrate or chemical process that involves enzymaticreactions.

Example 5

The present example relates to 50 ml of apple juice obtained by millingand filtering “golden” apples. Immediately after filtration the juicehas been divided into two samples and transferred into two sterilizedtransparent bottles and closed. In one of the two bottles ozone wasinsufflated before closing. The apple juice treated with ozone did notdarken but it maintained its original yellow-golden colour whereas thereference sample, not treated, turned quickly into brown. The fact thatozonized juice do not darken shows that the activity of enzymes thatcause the darkening of fruit juice (so called “polyphenol oxidase”, seeJ. J. Macheix et al. “Fruit Phenolics”, p. 296, CRC Press, 1990 BocaRaton, Fla.) have been inhibited by treatment with ozone. Treating withozone prevents also a growth of mold. The sample of juice not treatedwith ozone develops molds on the surface already after 4 days afterbottling whereas the sample treated with ozone does not develop any moldeven after 40 days from the bottling.

Example 6

Example 6 has been made on apple juice obtained by milling “golden”apple pulp and then filtrating it. Each experiment has been made on 25ml of apple juice in a 50 ml Pyrex vessel having a valve for vacuum. Thevacuum has been made with a water pump. The reference sample was simplytransferred and closed in the Pyrex vessel without any treatment. After4 days said sample developed mold, showed fermentation and remarkablydarkened. The sample of apple juice treated with ozone was prepared by aprocess comprising making vacuum in the vessel containing the juice andthen putting oxygen containing 10% ozone for a time of 20-30 seconds.During this time the apple juice was stirred and then evacuated again tothe pump for a second treatment similar to the previous. As expected,the introduction of ozone did not cause darkening and allowed the applejuice to remain light, and inhibited “polyphenol oxidase” enzymes. After4 days, but also after weeks, mold growth was not observed and smell andtaste remained good. Treating with ozone also inhibited completelyfermentation of the juice that instead was observed, as already said, inthe sample of reference. It is known that fermentation is an enzymaticreaction caused by yeasts. It is apparent that the ozone inhibitedcompletely the enzymatic functions of such micro-organisms acting thenalso as steriliser. It is also interesting to note other two points:First, owing to the short contact time, less than half of the ozonedelivered reacts with the substrate, as it is possible to measureiodometrically, whereas the remainder is recovered and used again.Second, another interesting aspect is that after 4 days, the juice iscompletely free from ozone, both free ozone and in the form ofperoxides, as verified with the KI test (absence of iodine as freeelement).

Example 7

A peach puree was prepared by milling a peach pulp. The puree obtainedwas divided into two 50 ml parts. One part was bottled as such, and onepart was bottled after treatment with ozone in the same way as describedin previous Example 6. Also in this case treating the puree with ozoneat bottling inhibits darkening of the puree due to action of “polyphenoloxidase” enzymes, avoiding also the growth of mold and fermentation ofthe puree, which instead were observed in the reference samples.

Example 8

Also surprising was the behaviour of the peach puree left in air in anot sterile environment. A peach puree (50 ml) was prepared milling apeach pulp and then was divided in two parts and left in two glasses inair at room temperature. One of the two samples was treated with a flowof ozone by means insulation in the mass. It is possible to observe, in24 hours time, even in air, that the treatment with ozone delays themold growth in the puree. On the contrary, the sample not treated withozone shows quick mold growth with large quantity of mold. Instead, inthe case of the sample treated with ozone, in addition to a delay ofseveral days in mold growth, such a growth was observed as very slow anddifficult.

Examples 5-8 have been carried out at room temperature and in an airatmosphere. Obviously, for particular applications in case of only UVirradiation it can be carried out in artificial reactive or not reactiveatmosphere around the substrate under irradiation.

The foregoing description of a specific embodiment will so fully revealthe invention according to the conceptual point of view, so that others,by applying current knowledge, will be able to modify and/or adapt forvarious applications such an embodiment without further research andwithout parting from the invention, and it is therefore to be understoodthat such adaptations and modifications will have to be considered asequivalent to the specific embodiment. The means and the materials torealise the different functions described herein could have a differentnature without, for this reason, departing from the field of theinvention. It is to be understood that the phraseology or terminologyemployed herein is for the purpose of description and not of limitation.

1. A process for inhibiting enzymatic activity in a substrate in liquidphase, in order to stop or inhibiting the course of enzymatic reactionscaused by enzymes present in said substrate, comprising the step ofdenaturing said enzymes, said denaturing step providing the applicationof denaturing means to said substrate up to a temperature set between 0and 60° C.
 2. The process of claim 1, wherein said denaturing step iscarried out at a temperature set between 10 and 50° C.
 3. The process ofclaim 1, wherein said denaturing step is carried out at roomtemperature.
 4. The process of claim 1, wherein said denaturing stepprovides the application to said substrate of gaseous ozone asdenaturing means.
 5. The process of claim 1, wherein said denaturingstep provides the application to said substrate of UV waves asdenaturing means for a predetermined time.
 6. The process of claim 1,wherein said denaturing step provides the application to said substrateof UV waves and of gaseous ozone as combined denaturing means.
 7. Theprocess of claim 6, wherein said combined denaturing means is generatedby a same device that is both a source of UV waves and an ozonegenerator.
 8. The process of claim 6, wherein the application to saidsubstrate of UV waves and of gaseous ozone as denaturing means iscarried out in different times.
 9. The process of claim 5, wherein thestep of application to said substrate of UV waves is carried out at apredetermined frequency and intensity at which said UV waves producesozone in said substrate.
 10. The process of claim 1, wherein saidsubstrate is selected from the group consisting of a food matrix, a pureenzyme or a mixture of enzymes in liquid phase, enzymes contained infruit juice or other liquid food in a real matrix, enzymes and mixtureof reagents used for biochemical synthesis, and waste material whereinhibiting the enzymatic activity is necessary before disposal.
 11. Theprocess of claim 1, wherein said food matrix is selected from the groupconsisting of vegetable juice, vegetable puree, fruit juice, and fruitpuree.